Download Isolation and Characterization of Foaming Proteins of

Isolation and Characterization of Foaming Proteins of Beer1
KATSUHIKO ASANO and NAOKI HASHIMOTO, The Research Laboratories of Kirin Brewery Co., Ltd., MiyaharaCho, Takasaki, Gumma Pref., 370-12 Japan
ABSTRACT
METHODS
"Foaming proteins" that retained the full foaming capacity of the original
beer were isolated by ultrafiltration followed by ammonium sulfate
precipitation and ion-exchange chromatography on diethylaminoethylcellulose. Because the content of foaming proteins correlated well with head
formation of many samples of beer, these foaming proteins seem to be
responsible for beer foaming. Foaming proteins consisted of three fractions
with molecular weights of 90,000-1,000,000, 40,000, and 15,000. These
three fractions were all surface-active but differed in the mechanism of their
contribution to foaming. The higher and medium molecular weight
fractions combined with isohumulones through «-amino groups to form
more surface-active complexes and thereby enhanced foaming. The lower
molecular weight fraction did not form these complexes appreciably,
probably because of its low content of t-amino groups. Immunological
studies showed that foaming proteins were formed primarily during
germination of barley. The level of foaming proteins decreased considerably
during brewing, particularly during kettle boiling; 160-620 mg/L of
foaming proteins survived in finished beer.
Key words: Beer, Brewing process, Foam, Isolation, Malting, Protein
Preparation of Samples from Beer
As shown in Fig. 1, 400 ml of unhopped beer was subjected to
ultrafiltration, using a Diaflo PM-30 ultrafiltration membrane,
and the retentate (Fraction 1) in the ultrafiltration cell was
saturated with ammonium sulfate at 0° C for one day. The resultant
precipitate (Fraction 3) was collected by filtration through filter
paper, redissolved in a minimum volume of deionized water,
dialyzed, and applied to a diethylaminoethyl-cellulose column (3.5
X 25 cm) that had been washed with 0.5M phosphate buffer, pH
8.0, and deionized water. The column was eluted first with 800 ml of
deionized water and then with 500 ml of 0.5M phosphate buffer,
pH 4.2. The effluent with phosphate buffer (Fraction 6) was
dialyzed and lyophilized.
Although some proteinaceous fractions of beer are generally
thought to be very important in beer foaming, these proteins have
not yet been fully characterized. Since Furnrohr (8) first suggested
the contribution of a proteinaceous fraction to beer foam in 1913,
many attempts have been made to isolate and characterize the
proteinaceous fractions responsible for head retention of beer.
Particularly during the last 10 years, the physical (11,13), chemical
(13), and immunological (13,15,18) properties of particular
proteinaceous fractions, such as "fraction X" (6), have been
elucidated, but the role of these proteinaceous fractions in beer
foaming is still unknown. Recently, S^rensen and Ottesen (32)
studied the fractionation of beer proteins in detail but were still
uncertain which fractions were responsible for foaming.
We succeeded in isolating the "foaming proteins" responsible for
foaming of beer. The present article describes the characterization
of these foaming proteins and gives clear evidence for their contribution to beer foaming.
' Presented at the 46th Annual Meeting, Minneapolis, MN, May 1980.
0361 -0470/80/04012909/S03.00/0
® 1980 American Society of Brewing Chemists, Inc.
Molecular Weight Estimation
By Gel Chromatography. The sample (2.5-4.5 mg) dissolved in
0.8 ml of O.OSMNaCl was applied to a Sephadex G-75 column (2 X
47 cm) equilibrated with 0.05M NaCl, and the column was eluted
with 0.05 Af NaCl at a flow rate of 20 ml/hr. Effluent was collected
in 4-ml fractions, and their protein and carbohydrate contents were
measured, respectively, by the methods of Lowry et al (22) and of
Molisch, as cited previously (7). The molecular weights of proteins
were estimated by comparison of their elution volumes with those
of standard proteins such as cytochrom c, myoglobin,
chymotrypsinogen A, ovalbumin, and bovine serum albumin.
The fraction eluted in the void volume from the Sephadex G-75
column was rechromatographed on a Sepharose 6B column (1.7
X 44 cm) equilibrated with 0.5M NaCl. The column was eluted with
0.5A/ NaCl at a flow rate of 20 ml/hr, and effluent was collected in
3.5-ml fractions and assayed for protein and carbohydrate as
described. The molecular weight of protein was estimated using
bovine serum albumin, y-globulin, and apo-ferritin as standard
proteins.
By Sodium Dodecyl Sulfate Gel Electrophoresis. The sample (4
mg) was dissolved in 0.5 ml of 0.01 M phosphate buffer, pH 7.2,
containing 1% sodium dodecyl sulfate (SDS) and 5% 2mercaptoethanol. The solution was incubated at room temperature
overnight, and then 10 n\ was applied to the top of a 10%
polyacrylamide gel (0.2 cm 2 X 9 cm) containing 0.1% SDS and
subjected to electrophoresis by the procedure of Weber and Osborn
130
Vol. 38 No. 4
(35). The gel was stained with Amido black and destained by
washing with 7% acetic acid. The mobility of the sample was
measured by scanning the gel at 477 nm with a Shimadzu CS-9IO
dual wavelength spectrodensitometer, and the molecular weight of
protein was estimated by comparison of its mobility with those of
standard proteins such as cytochrome c, chymotrypsinogen A,
ovalbumin, and bovine serum albumin.
Affinity Chromatography on Con A-Sepharose
The sample (4.7-25 mg) was dissolved in 1 ml of 0.01 Af Tris-HCl
buffer, pH 8.0, containing !%NaCl, ImMMgCh.and ImA/CaCh
and applied to a Con A-Sepharose CL-4B column (2 X 12.5 cm)
equilibrated with the same buffer. The column was first washed
with 75 ml of Tris-HCl buffer, and then the glycoprotein fraction
was eluted with 95 ml of the same buffer containing 0.05M amethyl-D-mannoside at a flow rate of 40 ml/hr. Effluent was
collected in 5-ml fractions, dialyzed, and analyzed for protein (22)
and carbohydrate (7).
Electrofocusing
Samples (0.1-0.4 mg) dissolved in 10-40 jul of deionized water
were applied to 5% polyacrylamide gel plates containing 2%
Beer
Degassed and ultrafiltered
with Diaflo PM-30 membrane
I
Retentate (Fraction 1)
Filtrate (Fraction 2)
Saturated with
I
Precipitate (Fraction 3)
I
Supernatant (Fraction 4)
Dialyzed and chromatographed
on DEAE-cellulose
Eluate with water
(Fraction 5)
Eluate with Phosphate
Buffer (Fraction 6)
Dialyzed and lyophilized
"Foaming Proteins"
Fig. 1. Procedure for isolation of "foaming proteins" from beer.
ampholytes (Ampholine PAG plate from LK.B), in a pH range
3.5-9.5, and electrofocusing was performed at 8° C for 3.5 hr at a
final voltage of 1,400 V and a final current of 10 mA. Proteins and
carbohydrates in the gel were stained with Coomassie Brilliant Blue
R-250 and periodic acid-Schiff reagent (12), respectively. The gel
was scanned at 560 nm for protein and at 535 nm for carbohydrate.
Immunoelectrophoresis
Soluble proteins were extracted from barley, malt, and rice by
the method of Grabar et al(10). The rabbit antisera towards these
soluble proteins of barley, malt, and rice—intact yeast cells and
foaming proteins—were prepared by Japan Immunoresearch
Laboratories Co., Ltd.
Immunoelectrophoresis was performed by the method of Grabar
and Williams (9) using 1% agarose gel in Tris-barbiturate buffer,
pH 8.6, at 8°C for 1 hr at 10 V/cm. After electrophoresis, the
trough of the gel was filled with antiserum, and the gel was left at
room temperature for 24 hr to allow immunodiffusion.
Immunoelectrophoresis at 8° C for 3 hr at 10 V/ cm was performed
by the method of Laurell (20), using 1% agarose gel in the same
buffer containing anti-foaming proteins serum. Protein
precipitates in the gel were stained with Coomassie Brilliant Blue
R-250.
Chemical Analyses
The protein and carbohydrate contents of samples were
determined by the micro-biuret method (17) and the phenol-sulfuric
acid method (16), using bovine serum albumin and arabinose,
respectively, as standards.
The amino acid composition of samples was analyzed with an
amino acid analyzer, JEOL model JLC-6AH, after the samples (5
mg) were hydrolyzed with 3 ml of 67V HC1 at 110° C for 22 hr in
evacuated sealed tubes.
Constituent sugars in samples were analyzed by the methods of
Sawardeker et al (29) after the samples (3.4-6.0 mg) were
hydrolyzed with 2 ml of Iff H2SOi at 100°C for 3 hr.
Measurement of Foam
Samples of 100-300 mg were dissolved in 1 L of 3.6% aqueous
ethanol at pH 4.2. Then 20 ml of the solution in a graduated test
tube (2.1X18 cm) was shaken up and down mechanically at 20° C
for 5 sec (400 times per minute, 4 cm amplitude). The volume of
the foam was then measured and recorded as the "head forming
capacity (ml)." The head forming capacity of degassed beer
measured in a similar way correlated well with head formation of
carbonated beer determined by the pouring method (3) (r = 0.91, n
= 12).
Preparation of Isohumulone
Isohumulones were extracted from Isolone (isomerized hop
extracts of Kalsec Co.) with isooctane and purified by silica gel
column chromatography (19).
Cfc
= 0.837
<
100 200 300 400 500 600 700
*
"FOAMING PROTEINS" IN BEER(mg/L)
Fig. 2. Correlation between the content of "foaming proteins" and head
forming capacity of lager beers. Values for head forming capacity were
corrected for variation of isohumulone content (3).
TABLE I
Head Forming Capacity of "Foaming Proteins"
Head Forming Capacity, ml
Concentration
At the Concentration
Beer Fraction
in Beer
At 100 mg/L
in Beer
(mg/L)
1
1.6
9,540
2
22,380
0.1
3
1,192
3.9
4
0.2
4,823
5
630
0
6
5.7
6.7
492
(Foaming proteins)
Original beer
6.9
ASBC Journal
131
RESULTS
Foaming Proteins from Beer
Table I shows the head foaming capacity of the beer fractions.
Fraction 6 contained most of the foam-enhancing substances and
had a head forming capacity equivalent to that of the original beer.
Because 67% of the material in Fraction 6 was protein, we named
this fraction "foaming proteins."
Figure 2 shows that the contents of foaming proteins ranging
from 160-620 mg/L in lager beer, correlated well with the head
forming capacities of these beers.
10
9
8
7
6
5
4
§
~ 0.5
E
O)
~ 0.4
O
Cytochrome c •
A
HIGHER MW FRACTION
VOID VOLUME
0.3
0.2
I o.
0.1
I
Chymo • trypsinogen A
• Myoglobin
Q
o
•Ovalbumin
\
Fractionation of Foaming Proteins
A solution of 100 mg of foaming proteins in 10 ml of 0.05M
ammonium formate was applied to a Sephadex G-75 column (5 X
54 cm) equilibrated with 0.05M ammonium formate. The column
was eluted with 0.05M ammonium formate at a flow rate of 55
ml/hr and effluent was collected in 15-ml fractions. Fractions of
effluent containing higher, medium, and lower molecular weight
materials, respectively, were combined as shown in Fig. 3, and
rechromatographed in the same fashion to yield 21.7,12.9, and 33.4
mg of lyophilized higher, medium, and lower molecular weight
materials, respectively.
Figures 4 and 5 show that the medium and lower molecular
weight fractions were almost homogeneous with respect to
molecular weight. The molecular weights of these two fractions
were estimated to be about 40,000 and 15,000 by gel
chromatography and 36,000 and 10,000 by SDS gel
electrophoresis. Figure 6 shows that the higher molecular weight
fraction was composed of at least three subfractions with molecular
weights of over 1,000,000, about 400,000, and 90,000, respectively.
Chemical Composition of Foaming Proteins
Table II shows that only 21% of the higher molecular weight
fraction was protein, whereas 75 and 65% of the medium and the
lower molecular weight fractions, respectively, were proteins. The
amino acid compositions of these three fractions were similar to
that of barley albumin or globulin (34). The higher and the medium
molecular weight fractions contained more lysine than did the
lower molecular weight fraction and did not contain cysteine and
•\Bovine Serum Albumin
MEDIUM MW FRACTION
MW 40,000
u
O
O
LOWER MW FRACTION
MW 15,000
£ 0.3
O
8 0.2
0.1
0.1
50
500
1000
ELUTION VOLUME (ml )
Fig. 3. Fractionation of "foaming proteins" by gel chromatography on
Sephadex G-75. • = protein, o = carbohydrate.
100
ELUTION VOLUME ( ml )
Fig 4. Estimation of molecular weight of higher, medium, and lower
molecular weight fractions of "foaming proteins" (3.1, 2.5, and 4.5 mg,
respectively), by gel chromatography on Sephadex G-75. • = protein, O =
carbohydrate.
132
Vol. 38 No. 4
methionine.
On the other hand, 64, 17, and 12% of the higher, medium, and
lower molecular weight fractions, respectively, consisted of the
carbohydrates arabionse, xylose, and glucose with small amounts
of mannose and galactose.
Figure 7 shows the isoelectric profiles of foaming proteins. The
medium and lower molecular weight fractions contained more than
ten protein species with isoelectric points of pH 4-5.5 and pH 3.5-5,
respectively. The higher molecular weight fraction also contained
at least six protein species with isoelectric points of pH 4-5.5.
Because some of the protein species stained with periodic acidSchiff reagent for carbohydrate, they seemed to be glycoproteins.
Con A-Sepharose chromatography (Fig. 8) showed that about 60
55, and 15% of the proteins in the higher, medium, and lower
molecular weight fractions, respectively, were glycoproteins.
Mechanism of Foaming of Foaming Proteins
Previously, we (3) found that foaming proteins combined
through their e-amino groups with isohumulones, and the resultant
surface-active complexes enhanced the foaming of beer. Figure 9
shows that a solution of the lower molecular weight fraction had
the highest surface activity and highest head forming capacity
of the three fractions. When isohumulones were added to the
solutions, the surface activities and head forming capacities of the
90-
Tt
O
60
50
Apo-ferritin«
40
h-
30
20
f-Globulin •
^^« Bovine
Bovin Serum Albumin
•xPvalbumin
Bovine
Serum Albumin*
O
Chymotrypsinogen A •,
Cytochrome
0.1
o o
o o
o
.MEDIUM
MW
o o
o o
O
FRACTION
o
O 9)
MW 36,000
O)
u
O 0.05
-I
0.4
1
LOWER
1
1
h
MW
H
1
H
FRACTION
MW 10,000
o
z
o
o
0.2
0.1 h
0
0.5
1
RELATIVE MOBILITY
Fig. S. Estimation of molecular weight of 5 ^g of "foaming proteins" by
sodium dodecyl sulfate gel electrophoresis. The higher molecular weight
fraction did not migrate in 10% polyacrylamide gel.
50
ELUTION VOLUME (nil)
Fig. 6. Estimation of molecular weight of 3 mg of the higher molecular
weight fraction of "foaming proteins"by gel chromatography on Sepharose
6B. • — protein, o ~ carbohydrate.
~ 2
ABSORBANCE AT 560 nm
ro
o
•< i
O
-i tn
05
§3
Q. p
S|
y I
II
00
n a
o o
-. C
< "•!5T
<£>
I TO'
2 - Si.w
CONCENTRATION (mg/ml)
•<" 9 3;»
|>f3
g C/l S. 3
3 n> o ~'
3 •a 3 •<
o 5" "" o
I
O
0 " g 1
5
1H >
^ 3='00
m
»' o 5 3
O
« u, 3
3
n PS.
o _,
0! ?'
3
.s,al
S w3 o°
a- o
3
-±
o
03
n
134
Vol. 38 No. 4
higher and medium molecular weight fractions increased greatly,
becoming more than that of the lower molecular weight fraction.
(Surface activity was determined as the difference in surface tension
of 3.6% aqueous ethanol before and after addition of foaming
proteins or of isohumulones.)
Table III shows that the head forming capacities of the higher
and medium molecular weight fractions were no longer enhanced
by addition of isohumulones when alkaline conditions prevented
isohumulones from combining with these fractions by suppressing
the dissociation of «-amino groups of the proteins.
The distinct difference in the foaming properties of the higher
and medium molecular weight fractions from that of the lower
molecular weight fraction led us to examine formation of
complexes between these three fractions and isohumulones,using
the method of dialysis equilibrium. As described in the previous
paper (3), 2 mg of each fraction dissolved in 2 ml of 0.1M phosphate
buffer containing 3.6% ethanol, pH 4.2 (inner solution) was put in a
cellophane tube and dialyzed against 0.8 mg of isohumulones in 4
ml of the same buffer (outer solution). When isohumulones diffuse
from the outer solution into the inner solution to combine with
foaming proteins, the concentration of uncombined isohumulones
in the inner solution decreases. Then more isohumulones diffuse
from the outer solution to the inner solution to restore the
TABLE II
Chemical Composition of "Foaming Proteins"
Molecular Weight Fraction
Higher Medium Lower
21
75
65
Protein content, %
Amino acid composition
of protein, mol %
6.1
9.2
Gly
7.5
6.7
8.6
7.4
Ala
4.1
6.4
5.5
Val
5.7
8.3
5.3
Leu
2.4
3.4
2.8
He
6.9
7.5
5.9
Ser
3.9
3.6
4.1
Thr
0
1.9
Cys
0
0
0
1.1
Met
4.1
2.2
1.5
Phe
0.9
1.1
1.7
Tyr
3.9
8.7
10.3
Pro
6.6
5.6
8.5
Asp
14.2
9.0
14.3
Glu
3.6
3.3
2.5
Lys
5.7
3.4
3.6
Arg
3.4
3.4
2.7
His
64
17
12
Carbohydrate content, %
Constituent sugars of
carbohydrate, mol %
48
48
18
Ara
19
35
25
Xyl
7
21
60
Glc
5
6
3
Man
5
trace
trace
Gal
TABLE III
Increase in Head Forming Capacity (ml) of "Foaming Proteins"
Caused by Isohumulones Under Acidic and Alkaline Conditions"
Molecular
PH
Weight
4.2
Fraction
11.0
Higher
+ 1.9
-0.2
Medium
+3.7
-0.9
Lower
+0.4
-1.3
'Average differences in head forming capacities of solutions of various
concentration before and after addition of 25 mg/ L of isohumulones.
equilibrium of isohumulone concentration. Table IV shows that
the concentration of isohumulones in the outer solution decreased
when the inner solution contained the higher or medium molecular
weight fraction but did not decrease appreciably when the inner
solution contained the lower molecular weight fraction. This result
suggests that isohumulones can combine with the higher and
medium molecular weight fractions.
Changes of Foaming Proteins during Malting and Brewing
Because foaming proteins formed immunoprecipitates with
antimalt serum, and to a lesser extent with antibarley and antiyeast
sera, as shown in Fig. 10, they seem to originate mainly from malt.
So the changes of foaming proteins during malting were examined
byimmunoelectrophoresis. Soluble protein fractions isolated from
5.7 mg of lyophilized germinating barley (Fuji nijo II) were
subjected to immunoelectrophoresis by the method of Laurell (20)
with 12.8 Ml/cm 2 of anti-foaming proteins serum. The content of
foaming proteins in the germinating barleys was determined using a
calibration curve constructed by immunoelectrophoresis of known
amounts of foaming proteins with anti-foaming proteins serum.
Protease activities of the germinating barleys were determined by
the method of Miller (23). Barley germinated for six days was
kilned and the resultant malt was used for preparation of Congress
wort.
Figure 11 shows that the levels of foaming proteins in the barley
increased rapidly with increase in the protease activity of
germinating barley, reaching a maximum on the fourth day of
_
J
'.
HIGHER MW
FRACTION
> 10
o
z
O
_.
t
2
u
•
o •*
MEDIUM MW
FRACTION
..O
/
° -/*
•"^
LOWER MW
FRACTION
JO
/
*^
A
o
/•
^J*
su
O^*
c
>>
®/
0
•D
*
>
1—
o
LU
P
o o <
UJ
I
U
A---A"
A^^
x^
A''
,wft
^
A
A
/
UJ
A
A^x
^i— ~"ix
"
^**
_A-'
' 10 <
A''
I
I
I
i
l
l
'
5 U
5E
ce
D
n (/)
CONCENTRATION OF "FOAMING PROTEINS" (mg/L)
Fig. 9. Head forming capacity (•) and surface activity (A) of 100-300 mg of
"foaming proteins" dissolved in 1L of 3.6% aqueous ethanol, pH 4.2. After
25 mg of isohumulones were added, the solutions were retested (o and A,
respectively).
TABLE IV
Combination of Isohumulones with "Foaming Proteins"
Isohumulones in Outer Solution
Decrease in
Concentration
Concentration
After Dialysis
After Dialysis"
Inner Solution
(mg/L)
(mg/L)
Without "foaming proteins"
121.8
With "foaming proteins"
molecular weight fraction
Higher
117.2
4.2
Medium
115.2
6.6
Lower
120.8
1.0
"Difference between the blank and the sample.
ASBC Journal
germination and then decreasing gradually in the latter period of
germination.
Figure 12 shows the changes of foaming proteins during brewing.
Foaming proteins of wort and fermenting wort were isolated by the
procedure used for their isolation from beer. The levels of foaming
proteins in wort decreased during brewing, particularly during
kettle boiling, and only half the total foaming proteins in sweet wort
survived in finished beer. M ost of the less acidic species of foaming
proteins with isoelectric points of pH 4.3-5.5 were lost during kettle
boiling (Fig. 13).
Figure 14 shows the effects of process variables on the levels of
foaming proteins. Beer brewed from under-modified malt
(Kolbach index = 39.9%) retained about 30% more foaming
proteins than did beer brewed from control malt (Kolbach index =
43.7%). A short protein-rest in mashing also resulted in an increase
of about 10% more foaming proteins in finished beer. When the
wort was boiled with hops, the levels of foaming proteins in the
resultant beer was 30% less than that in unhopped beer. Similarly,
the accelerated decrease of foaming proteins in the wort was caused
by boiling with humulones. Therefore, not only polyphenols
derived from hops but also humulones and isohumulones seem to
accelerate the precipitation of foaming proteins during kettle
boiling. Reduction of the boiling time of wort from 90 to 30 min
reduced this precipitation or coagulation of foaming proteins. The
less acidic species of foaming proteins with isoelectric points of pH
4.3-5.5 were less stable during the brewing process (Fig. 15).
135
DISCUSSION
Although many attempts have been made (1,2,4-6,11,21,24-28,
30,33,36), until now foam-enhancing proteins have not been isolated from beer. In the present work, foaming proteins that retained
the full foaming capacity of the original beer were isolated and
purified. The contents of foaming proteins correlated well with
SWEET WORT
HOPPED WORT
50
100
ELUTION VOLUME ( m l )
Fig. 12. Changes during brewing of "foaming proteins" isolated from 30 ml
of worts and beers, chromatographed on Sephadex G-75. •, O , A, o =
protein;
,
, —,
= carbohydrate.
ANTI-BARLEY SERUM
1.5
ANTI-MALT SERUM
SWEET WORT
ANTI-RICE SERUM
,.,.,^ ANTI-YEAST SERUM
Fig. 10. Immunoelectrophoretic analysis of 100 pg of "foaming proteins"
that were allowed to react with lOOjulof antibarley, antimalt, antirice, and
antiyeast sera.
E
c
o
in
1.0
5
LLJ
o
I
O
I
o
CO
o:
0.5
8CO
a.
O
- 0.5
O
z <
o
o
O u.
>
UJ
0
2
4
6
GERMINATION
H- H-l-
o
OL
Q.
PERIOD (day)
Fig. 11. Changes of "foaming proteins" during malting. The protease
activity of the malt is represented as 1 unit.
4
5
6
7
8
9
ISOELECTRIC POINT (pH)
Fig. 13. Changes during brewing of isoelectric profiles of "foaming
proteins" isolated from 0.5 ml of worts and beers. The dotted peaks
represent less acidic proteins species.
136
Vol. 38 No. 4
head formation of many samples of beer.
Recently, Schulze et al (31) reported that some proteinaceous
fractions concentrating in beer foam were composed of three
fractions with molecular weights of 150,000, 90,000, and 10,000,
and Hejgaard and S^rensen (13) suggested that barley protein Z
(14), with a molecular weight of 40,000, was concentrated in beer
PROTEIN-REST
MALT MODIFICATION
0.3
0.2
60C-15min
Kl = 39.9%
/Kl=43.7 %
0.1
o
HOPS
KETTLE BOILING
ct
^
z 0.3
UJ
30min
90 min
O
o
0.2
0.1
100
50
100
ELUTION VOLUME (ml)
Fig. 14. Effects of process variables on the levels of "foaming proteins"
isolated from 30 ml of beers under various conditions, chromatographed on
Sephadex G-75. •, o, A = protein;
,
, — = carbohydrate.
50
4
5
6
7
8
9
4
5
6
7
8
9
ISOELECTRIC POINT (pH)
Fig. 15. Effects of process variables on the isoelectric profiles of "foaming
proteins" isolated from 0.5 ml of beers brewed under various conditions.
foam. Our foaming proteins were also composed of three fractions
with molecular weights of 90,000-1,000,000, 40,000, and 15,000.
In general, proteins with molecular weights of 10,000-100,000 or
more seem to participate in head formation of beer.
The mechanisms by which these three fractions of foaming
proteins contribute to foaming of beer differed. The higher and
medium molecular weight fractions combined with isohumulones
to form more surface-active complexes and thereby enhanced the
foaming, whereas the lower molecular weight fraction did not form
these complexes appreciably. Because «-amino groups of lysine
residues in foaming proteins combine electrostatically with acidic
groups of isohumulone molecules, as reported previously (3), the
higher and medium molecular weight fractions, which contain
more e-amino groups than does the lower molecular weight
fraction, must combine preferentially with isohumulones.
Accordingly, both foaming proteins derived from malt and
isohumulones derived from hops are essential for head formation
of beer. Foaming proteins, particularly the higher and medium
molecular weight fractions, combine with isohumulones, probably
in the surface of small bubbles in beer (3), and the resultant surfaceactive complexes contribute to the foaming of beer.
By immunological studies, we obtained clear evidence that
foaming proteins are formed in germinating barley. Precursors of
foaming proteins, present in barley in insoluble form, are probably
solubilized to foaming proteins by a protease activated in
germinating barley. During further germination, the solubilized
foaming proteins seem to be degraded enzymatically. Foaming
proteins derived from malt are also degraded enzymatically during
mashing. Therefore, under-modification of malt and brief mashing
are beneficial for head formation of the resultant beer.
ACKNOWLEDGMENTS
We wish to thank the management of Kirin Brewery Co., Ltd. for
permission to publish this work. We are grateful for the continuous
encouragement of Y. Kuroiwa, Senior Managing Director, and Y. Horie,
the former director of the Research Laboratories.
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[Received May 12, 1980]